This invention relates to mixing fluids, and more particularly relates to an apparatus and method for mixing a small quantity of fluid within a hybridization chamber.
Conventional methods for mixing relatively large volumes of fluids utilize mixing devices that mix the fluids by shaking the container, by a rapid mechanical up and down motion, or by a rocking motion that tilts the container back and forth. Conventional mixing methods, however, cannot normally be applied to chemical and biological assays involving small samples in a fluid because the capillary strength of the containment system often exceeds the forces generated by shaking or rocking, thereby preventing or minimizing motion of the fluid. As an illustration, a small amount of fluid sample, when spread across a large reactive surface, will form a thin film of fluid that may have a thickness of a few microns to a few millimeters. In such situations, the fluid may not adequately contact the entire reactive surface or the reactive compounds in the fluid may be very dilute, thereby resulting in a reaction that is limited by the rate of diffusion through the fluid. Inadequate mixing can adversely affect the reproducibility of results, the sensitivity or specificity of the reaction, the rate of reaction, the extent of reaction, the homogeneity, or the percent yield.
Inadequate mixing is a particular problem in chemical and biological assays performed on microarrays that typically involve very small samples of chemical, biochemical, or biological fluids. In microarrays, the targets or probes are usually immobilized on a solid support having a surface area that can be smaller than a few square centimeters. The solid support is typically a glass or fused silica slide which has been treated to facilitate attachment of either the targets or probes. A liquid containing the reactants is then brought into contact with the immobilized nucleotides on the solid support. In a typical procedure, the fluid is placed on the support, and the fluid and solid support are covered with another slide and placed in an environmentally controlled chamber such as an incubator for several hours. Typically, the reactants in the liquid diffuse to the interface where they react with the immobilized probes or targets, and a reaction, such as hybridization reaction, occurs. Detection of hybridization between an immobilized nucleotide sequence and a complementary probe offers a convenient and reliable means for isolating, sequencing, and analyzing nucleotides. However, when diffusion is the only means of bringing the reactants in the liquid in contact with the immobilized nucleotides, problems encountered include poor hybridization kinetics and poor reaction specificity and sensitivity.
The present invention describes an apparatus and method for mixing of a film of fluid by moving a bubble within the film. The use of bubbles for mixing large volumes of liquids is well known. For example, U.S. Pat. No. 5,443,985 to Lu et al. and U.S. Pat. No. 5,605,653 to DeVos describe the mixing and aeration of large volumes of liquid, such as a culture medium in a cell culture bioreactor by introducing extraneous gas at the bottom of the reactor thereby creating bubbles that travel upwards, thus mixing the liquid medium.
The invention, in one embodiment, is an apparatus for mixing a film of fluid, particularly a chemical, biochemical, or biological fluid, which typically comprises a reaction mixture, the apparatus comprising a first substrate having an inner surface and a substantially parallel second substrate having an inner surface, the inner surfaces defining a closed chamber therebetween. The closed chamber is adapted to retain a quantity of fluid so that the fluid is in contact with both inner surfaces. In addition, the apparatus comprises a means for introducing a bubble in the fluid, and a means for moving the bubble in the fluid. The inner surface of one or both of the substrates is functionalized with reactive moieties that can react with the components contained in the fluid.
The invention also provides a method for mixing a film of fluid comprising providing a first substrate and a substantially parallel second substrate, each substrate having an inner surface, the inner surfaces defining a closed chamber therebetween. The closed chamber is adapted to retain a quantity of fluid so that the fluid is in contact with both inner surfaces. The method further comprises introducing a fluid containing a plurality of components into the closed chamber so as to provide a film of fluid therein, introducing a bubble within the fluid film, and a means for moving the bubble in the film of fluid thereby mixing the film of fluid.